Cationic transport reagents

ABSTRACT

For use in transporting biologically active species into and through membrane barriers, a symmetrical cationic diamine compound having the general structure ##STR1## wherein m=1-10; R 1  is a hydrogen, an alkyl group, an alkenyl group, a hydroxylated alkyl or alkenyl group, or an ether containing alkyl or alkenyl group; R 2  is an alkyl group, an alkenyl group, or an alkyl or alkenyl containing acyl group; R 3  is a hydrogen, an alkyl group, an alkenyl group, a hydroxylated alkyl or alkenyl group, or an ether containing alkyl or alkenyl group; R 4  is a hydrogen, an alkyl group, an alkenyl group, a hydroxylated alkyl or alkenyl group, or an ether containing alkyl or alkenyl group; and X -  is an anion.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of application Ser. No. 08/316,719 filedon Sep. 30, 1994, now U.S. Pat. No. 5,527,928.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Provided are cationic lipids that bind and transport polynucleotides,polypeptides, pharmaceutical substances and other biologically activespecies through membrane barriers. More specifically, symmetricaldiamine cationic lipids are disclosed that complex with selectedmolecular species and facilitate delivery of those selected species intoand through membranes and comparable boundary structures.

2. Description of the Background Art

Cellular transfection strategies for gene therapy and similar goals havebeen designed and performed, but many of these procedures involverecombinant virus vectors and various problems exist with these viralgene transfer systems. Even generally advantageous adenovirus techniquesencounter difficulties since most humans have antibodies to many of theadenovirus serogroups, including those that have been chosen as vectors.Wild type adenoviral superinfection of an adenoviral vector treatedpatient may result in propagating the recombinant vector as a defectiveviral particle, with the ability to infect many unintended individuals(if chosen to have a rare serogroup). The chance of adenoviralcontamination is quite low but not impossible. The safety of using thesegenetic materials in humans remains unclear and thus hazardous.

Safe, non-viral vector methods for transfection or gene therapy areessential. A few such safe lipid delivery systems for transporting DNA,proteins, and other chemical materials across membrane boundaries havebeen synthesized by research groups and business entities. Most of thesynthesis schemes are relatively complex and generate transportershaving only limited transfection abilities. A need exists in the fieldof cationic lipid transporters for cationic species that have a highbiopolymer transport efficiency. It has been known for some time thatquaternary ammonium derivatized (cationic) liposomes spontaneouslyassociate with DNA, fuse with cell membranes, and deliver the DNA intothe cytoplasm. LIPOFECTIN™ represents a first generation of cationicliposome formulation development. LIPOFECTIN™ is composed of a 1:1formulation of the quaternary ammonium containing compound DOTMA anddioleoylphosphatidylethanolamine sonicated into small unilamellarvesicles in water. One problem with LIPOFECTIN™ is that it containsnon-metabolizable ether bonds. Other problems with LIPOFECTIN™ are aninhibition of protein kinase C activity and direct cytotoxicity. Inresponse to these problems, a number of other related compounds havebeen developed. The diamine compounds of the subject invention improveupon the capabilities of existing cationic transporters and serve asvery efficient delivery means for biologically active chemicals.

As indicated immediately above, various cationic lipids have beensynthesized in previous references. For example, U.S. Pat. No. 4,812,449discloses in situ active compound assembly of biologically active agentsat target locations in preference to surroundings which are desired tobe unaffected. Several charged and uncharged amine derivatives aredescribed.

Introduced in U.S. Pat. No. 5,171,678 are lipopolyamines and their usefor transfecting eukaryotic cells. A polynucleotide is mixed with thesubject lipopolyamine and contacted with the cells to be treated.

U.S. Pat. Nos. 5,186,923 and 5,277,897 relate an enhancement of cellularaccumulation of lipophilic cationic organometallic compounds byreduction of the intramembrane potential. Technetium containingcompounds are disclosed.

Lipophilic cationic compounds are presented in U.S. Pat. No. 5,208,036.Asymmetrical amine compounds are synthesized and employed in a methodfor DNA transfection.

U.S. Pat. No. 5,264,618 discloses cationic lipids for intracellulardelivery of biologically active molecules. Asymmetric ammoniumcontaining cationic lipids are presented for transporting molecules intomembranes enclosed systems.

Transfection of nucleic acids into animal cells via a neutral lipid anda cationic lipid is revealed in U.S. Pat. No. 5,279,833. Liposomes withnucleic acid transfection activity are formed from the neutral lipid andthe ammonium salt containing cationic lipid.

U.S. Pat. No. 5,334,761 describes other amine containing cationic lipidsare reported. Cationic lipids are utilized to form aggregates fordelivery of macromolecules and other compounds into cells.

The foregoing patents reflect the state of the art of which theapplicants are aware and are tendered with the view toward dischargingapplicants' acknowledged duty of candor in disclosing information whichmay be pertinent in the examination of this application. It isrespectfully submitted, however, that none of these patents teach orrender obvious, singly or when considered in combination, applicants'claimed invention.

SUMMARY OF THE INVENTION

An object of the present invention is to disclose a category of diaminesthat greatly facilitate the delivery of biologically active compoundsthrough membrane structures.

Another object of the present invention is to present a group ofsymmetrical diamine cationic compounds that assist in the transport ofselected macromolecules and other substances into and past membranebarriers.

A further object of the present invention is to relate a collection ofbiologically active molecule transporters having easily variable lipidcomponents linked to a symmetrical polyhydroxyl containing diamine corestructure.

Disclosed are novel diamine cationic transporter molecules thatfacilitate the delivery of such compounds as polynucleotides,polypeptides, and the like into and beyond membrane walls. Generallyrelated are symmetrically structured cationic diamines, eitherpolyhydroxylated or otherwise quaternized, having at least a pair ofidentical lipoyl moieties selected from a group consisting of an alkylchain, an alkenyl chain, and an alkyl or alkenyl containing acyl chain.More specifically, a compound having the structure: ##STR2## whereinm=1-10; R₁ is a hydrogen, an alkyl group, an alkenyl group, or ahydroxylated alkyl or alkenyl group; R₂ is an alkyl group, an alkenylgroup, or an alkyl or alkenyl containing acyl group; R₃ is a hydrogen,an alkyl group, an alkenyl group, or a hydroxylated alkyl or alkenylgroup; R₄ is a hydrogen, an alkyl group, an alkenyl group, ahydroxylated alkyl or alkenyl group, or an ether containing group; andX⁻ is an anion.

In particular, preferred compositions areN,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediaminiumiodide, given the nickname PolyGum in view of its binding affinity,O-alkylated derivatives of PolyGum,N,N'-dimethyl-N,N,N',N'-tetra(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediaminiumiodide, and O-alkylated derivatives ofN,N'-dimethyl-N,N,N',N'-tetra(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediaminiumiodide. Even though these are preferred compositions, the length anddouble bond characteristics of the R₂ group (as is detailed below) andthe presence or absence of a carbonyl in the R₂ group is variable.

Other objects, advantages, and novel features of the present inventionwill become apparent from the detailed description that follows, whenconsidered in conjunction with the associated drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that demonstrates the effectiveness of a 50:50 mixtureof DOPE:PolyGum with DNA, compared with DNA only, in transfecting NIH3T3 cells in a serum-free environment.

FIG. 2 is a graph that illustrates that liposome structure (SV versusMLV forms) influences the efficiency of transfecting NIH 3T3 cells.

FIG. 3 is a graph showing the effects of serum on PolyGum (MLV) mediatedtransfection of NIH 3T3 cells.

FIG. 4 is a graph depicting the influence of phosphatidylethanolamine(PE) side chain structure on transfection of NIH 3T3 cells using a 50:50mixture of the PE derivatives and PolyGum in the presence of 10% calfserum and without serum.

FIG. 5 is a graph showing the optimization of the mole ratio of DOPE toPolyGum (SV) in serum-free transfection of NIH 3T3 cells.

FIG. 6 is a graph illustrating the optimization of the charge ratio ofPolyGum (SV) to DNA in serum-free transfection of NIH 3T3 cells.

FIG. 7 is a graph portraying an efficiency comparison of utilizedcationic lipids in the serum-free transfection of DU-145 cells.

FIG. 8 is a graph showing an efficiency comparison of utilized cationiclipids in transfection of DU-145 cells in the presence of 2% FBS.

FIG. 9 is a graph showing a hydrophobic and polar domain transfectionanalysis of PolyGum and PolyGum methoxy derivatives.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring now to the following disclosure and to the data presented inFIGS. 1-9, there is described a preferred embodiment of a symmetricalcationic diamine having at least a pair of identical lipoyl moietiesselected from a group consisting of an alkyl chain, an alkenyl chain,and an alkyl or alkenyl containing acyl chain.

Generally, the diamine is polyhydroxylated and has a generalizedstructure of: ##STR3## wherein m=1-10, preferably 1; R₁ is a hydrogen,an alkyl group, an alkenyl group, or a hydroxylated alkyl or alkenylgroup; R₂ is an alkyl group, an alkenyl group, or an alkyl or alkenylcontaining acyl group; R₃ is a hydrogen, an alkyl group, an alkenylgroup, or a hydroxylated alkyl or alkenyl group; R₄ is a hydrogen, analkyl group, an alkenyl group, a hydroxylated alkyl or alkenyl group, oran ether containing group; and X⁻ is an anion. The extra, with m morethan 1, number of methylenes is introduced by standard procedures thatcomplement the described subject synthetic pathways.

More specifically, one preferred structure is: ##STR4## wherein forcompound A: n=0-10, usually between 0 and 3, preferably 1; R₁ is ahydrogen, an alkyl group, an alkenyl group, or a hydroxylated alkyl oralkenyl group, generally having from 1 to 10 carbons, preferably 1carbon; R₂ is an alkyl group, an alkenyl group, or an alkyl or alkenylcontaining acyl group; R₃ is a hydrogen, an alkyl group, an alkenylgroup, or a hydroxylated alkyl or alkenyl group, often an alkyl group offrom 1 to 10 carbons, preferably a methyl group; and X⁻ is an anion,usually a halide, and preferably iodide.

A second preferred structure is: ##STR5## wherein for compound B:n=0-10, usually between 0 and 3, preferably 1; R₂ is an alkyl group, analkenyl group, or an alkyl or alkenyl containing acyl group; R₃ is ahydrogen, an alkyl group, an alkenyl group, or a hydroxylated alkyl oralkenyl group, often an alkyl group of from 1 to 10 carbons, preferablya methyl group; and X⁻ is an anion, usually a halide, and preferablyiodide.

A third preferred structure is: ##STR6## wherein for compound C: n=0-10,usually between 0 and 3, preferably 1; R₁ is a hydrogen, an alkyl group,an alkenyl group, or a hydroxylated alkyl or alkenyl group, generallyhaving from 1 to 10 carbons, preferably 1 carbon; R₂ is an alkyl group,an alkenyl group, or an alkyl or alkenyl containing acyl group; R₃ is ahydrogen, an alkyl group, an alkenyl group, or a hydroxylated alkyl oralkenyl group, often an alkyl group of from 1 to 10 carbons, preferablya methyl group; R₅ is an alkyl or alkenyl group of from 1 to 10 carbons,preferably a methyl group; and X⁻ is an anion, usually a halide, andpreferably iodide.

A fourth preferred structure is: ##STR7## wherein for compound D:n=0-10, usually between 0 and 3, preferably 1; R₂ is an alkyl group, analkenyl group, or an alkyl or alkenyl containing acyl group; R₃ is ahydrogen, an alkyl group, an alkenyl group, or a hydroxylated alkyl oralkenyl group, often an alkyl group of from 1 to 10 carbons, preferablya methyl group; R₅ is an alkyl or alkenyl group of from 1 to 10 carbons,preferably a methyl group; R₆ is an alkyl or alkenyl group of from 1 to10 carbons, preferably a methyl group; and X⁻ is an anion, usually ahalide, and preferably iodide.

To facilitate discussing the subject compounds, a list of abbreviations,nicknames, or acronyms follows:

    ______________________________________    DC Cholesterol                 3β- N-(N',N'-dimethylaminoethane)-carbamoyl!                 cholesterol    DCPE         Dicaproylphosphatidylethanolamine    DMAP         4-(N,N-dimethylamino)pyridine    DMEM         Dulbecco's modified Eagles medium    DMPE         Dimyristoylphosphatidylethanolamine    DOGS         Dioctadecylamidoglycyl spermidine    DOHME        N- 1-(2,3-dioleoyloxy)propyl!-N- 1-(2-                 hydroxyethyl)!-N,N-dimethylammonium iodide    DOPE         Dioleoylphosphatidylethanolamine    DOSPA        2,3-Dioleoyloxy-N- 2-                 (sperminecarboxamido)ethyl!-N,N-dimethyl-1-                 propanaminium trifluoroacetate    DOTAP        N- 1-(2,3-dioleoyloxy)propyl!-N,N,N-                 trimethylammonium iodide  DIESTER!                 (Boehringer Mannheim GmbH)    DOTMA        N- 1-(2,3-dioleyloxy)propyl!-N,N,N-                 trimethylammonium bromide  DIETHER!    DSPE         Distearoylphosphatidylethanolamine    DU-145       Human prostatic carcinoma cells for a                 representative human tumor cell line    FBS          Fetal Bovine Serum    Lipofectamine                 DOSPA + DOPE    Lipofectin Reagent                 DOTMA + DOPE (Vical Inc.)    NIH 3T3      Murine fibroblast cells for a representative                 human cell line    MLV          Multilamellar vesicles    PE           Phosphatidylethanolamine    PolyGum (DO-PG-OH)                 N,N,N',N'-tetramethyl-N,N'-bis(2-                 hydroxyethyl)-2,3-di(oleoyloxy)-1,4-                 butanediaminium iodide    SV           Sonicated or extruded vesicles    Transfectam Reagent                 DOGS    DM-PG-OH     PolyGum with palmitoyl in place of oleoyl    DO-PG-Me     Methoxy derivative of PolyGum    DP-PG-Me     Methoxy derivative of PolyGum with palmitoyl                 in place of oleoyl    DM-PG-Me     Methoxy derivative of PolyGum with myristoyl                 in place of oleoyl    DL-PG-Me     Methoxy derivative of PolyGum with lauroyl in                 place of oleoyl    ______________________________________

Although other possible methods of synthesizing the subject compoundsare possible, preferred and general synthetic schemes for cationicdiamine compounds are: ##STR8## where: n=1-10; R₁ is a hydrogen, analkyl group, an alkenyl group, or a hydroxylated alkyl or alkenyl group;R₂ is an alkyl group, an alkenyl group, or an alkyl or alkenylcontaining acyl group; R₃ is a hydrogen, an alkyl group, an alkenylgroup, or a hydroxylated alkyl or alkenyl group; and X⁻ is a halide.

In the general synthesis scheme for Compound A the first step involvesreacting a tert-butyldiphenylsilyloxy derivatized material (made via areaction of the hydroxyethyl starting material with ClSiPh₂ tBu) with1,3-butane diepoxide in the presence of lithium perchlorate in absoluteethanol. The second step is a reaction with an alkyl or alkenyl halideor an alkyl or alkenyl containing acyl halide. The third step istetrabutylammonium fluoride and THF initiated removal of thetert-butyldiphenylsilyloxy protection groups to produce the generalprecursor compound. The general precursor compound is then allowed toreact with a selected alkyl, alkenyl, or hydroxylated alkyl or alkenylhalide. ##STR9## where: n=1-10; R₁ is a hydrogen, an alkyl group, analkenyl group, or a hydroxylated alkyl or alkenyl group; R₂ is an alkylgroup, an alkenyl group, or an alkyl or alkenyl containing acyl group;R₃ is a hydrogen, an alkyl group, an alkenyl group, or a hydroxylatedalkyl or alkenyl group; and X⁻ is a halide.

In the general synthesis scheme for Compound B the first step involvesreacting a tert-butyldiphenylsilyloxy derivatized material (made via areaction of the dihydroxyethyl starting material with ClSiPh₂ tBu) with1,3-butane diepoxide in the presence of lithium perchlorate in absoluteethanol. The second step is a reaction with an alkyl or alkenyl halideor an alkyl or alkenyl containing acyl halide. The third step istetrabutylammonium fluoride and THF initiated removal of thetert-butyldiphenylsilyloxy protection groups to produce the generalprecursor compound. The general precursor compound is then allowed toreact with a selected alkyl, alkenyl, or hydroxylated alkyl or alkenylhalide. ##STR10## where: n=1-10; R₁ is a hydrogen, an alkyl group, analkenyl group, or a hydroxylated alkyl or alkenyl group; R₂ is an alkylgroup, an alkenyl group, or an alkyl or alkenyl containing acyl group;R₃ is a hydrogen, an alkyl group, an alkenyl group, or a hydroxylatedalkyl or alkenyl group; R₅ is an alkyl or alkenyl group of from 1 to 10carbons, preferably a methyl group; and X⁻ is a halide.

In the general synthesis scheme for Compound C the first step involvesreacting an ether containing starting material with 1,3-butane diepoxidein the presence of lithium perchlorate in absolute ethanol. The secondstep is a reaction with an alkyl or alkenyl halide or an alkyl oralkenyl containing acyl halide. The general precursor compound is thenallowed to react with a selected alkyl, alkenyl, or hydroxylated alkylor alkenyl halide. ##STR11## where: n=1-10; R₂ is an alkyl group, analkenyl group, or an alkyl or alkenyl containing acyl group; R₃ is ahydrogen, an alkyl group, an alkenyl group, or a hydroxylated alkyl oralkenyl group; R₅ is an alkyl or alkenyl group of from 1 to 10 carbons,preferably a methyl group; R₆ is an alkyl or alkenyl group of from 1 to10 carbons, preferably a methyl group; and X⁻ is a halide.

In the general synthesis scheme for Compound D the first step involvesreacting a diether containing starting material with 1,3-butanediepoxide in the presence of lithium perchlorate in absolute ethanol.The second step is a reaction with an alkyl or alkenyl halide or analkyl or alkenyl containing acyl halide. The general precursor compoundis then allowed to react with a selected alkyl, alkenyl, or hydroxylatedalkyl or alkenyl halide.

A preferred subject composition has the structure: ##STR12## wherein Ris an alkyl or alkenyl group, preferably --CH₂ (CH₂)₆ CH═CH(CH₂)₇ CH₃,--(CH₂)₁₄ CH₃, --(CH₂)₁₂ CH₃, or --(CH₂)₁₀ CH₃ and X⁻ is an anion,preferably a halide such as iodide.

A more preferred Compound A species of the subject invention has thenameN,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediaminiumiodide (PolyGum) with the following structure: ##STR13##

A synthesis scheme for the preferred compound is as follows: ##STR14##

A second preferred composition has the structure: ##STR15## wherein R isan alkyl or alkenyl group, preferably --CH₂ (CH₂)₆ CH═CH(CH₂)₇ CH₃,--(CH₂)₁₄ CH₃, --(CH₂)₁₂ CH₃, or --(CH₂)₁₀ CH₃ and X⁻ is an anion,preferably a halide such as iodide.

In particular, one Compound B species of the subject invention has thenameN,N'-dimethyl-N,N,N',N'-tetra(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediaminiumiodide with the following structure: ##STR16##

A synthesis scheme for the generally preferred Compound B species is asfollows: ##STR17##

A third preferred composition has the structure: ##STR18## wherein R isan alkyl or alkenyl group, preferably --CH₂ (CH₂)₆ CH═CH(CH₂)₇ CH₃,--(CH₂)₁₄ CH₃, --(CH₂)₁₂ CH₃, or --(CH₂)₁₀ CH₃ and X⁻ is an anion,preferably a halide such as iodide.

In particular, one preferred Compound C species of the subject inventionhas the nameN,N,N',N'-tetramethyl-N,N'-bis(2-methoxyethyl)-2,3-di(oleoyloxy)-1,4-butanediaminiumiodide with the following structure: ##STR19##

A synthesis scheme for the generally preferred Compound C species is asfollows: ##STR20##

A fourth preferred composition has the structure: ##STR21## wherein R isan alkyl or alkenyl group, preferably --CH₂ (CH₂)₆ CH═CH(CH₂)₇ CH₃,--(CH₂)₁₄ CH₃, --(CH₂)₁₂ CH₃, or --(CH₂)₁₀ CH₃ and X⁻ is an anion,preferably a halide such as iodide.

A fourth more preferred Compound D species of the subject invention hasthe nameN,N'-dimethyl-N,N,N',N'-tetra(2-methoxyethyl)-2,3-di(oleoyloxy)-1,4-butanediaminiumiodide with the following structure ##STR22##

A synthesis scheme for the generally preferred Compound D species is asfollows: ##STR23##

Rationale for Variations in Hydrophobic Domain

As seen in preferred compound, PolyGum, the long lipid tails are botholeoyl groups, however, other lipid tails are acceptable and within therealm of this disclosure. A study (Balasubramaniam, R. P., Bennett, M.J., Gruenert, D., Malone, R. W., and Nantz, M. H., "Hydrophobic Domainof Cationic Lipids Influence Respiratory Epithelial Cell DNATransfection" manuscript in preparation, which is herein incorporated byreference) involving cationic lipids, which contain aN,N-dimethyl-N-(2-hydroxyethyl)ammonium group (CH₃)₂ (HOCH₂ CH₂ --)N⁺--R! as the hydrophilic domain (polar head group component present inPolyGum) and which contain various fatty acid combinations to comprisethe hydrophobic domain, has shown that subtle changes in the compositionof the hydrophobic domain do affect the performance of these lipids asmediators of polynucleotide delivery into mammalian cells(transfection). However, in all examples, the cationic lipids showedactivity as agents for polynucleotide transfection. Therefore, thevarious combinations of fatty acid side chains represent only analogouschanges in the overall structure of the cationic lipid, and in each casethe cationic lipid is apt to demonstrate transfection activity.

The derivatization of DOHME (a cationic asymmetric lipid containing amono-ammonium head group) involving changes in the hydrophobic domainhas led to the discovery that all the derivatives display transfectionactivity, yet in varying amounts. By analogy, changes in the hydrophobicdomain of PolyGum will lead to new lipids which possess transfectionactivity. Additionally, this expectation is supported by recentliterature work (Felgner, P. L. et al J. Biological Chem. 1994, 269,2550) which demonstrates that changes in the hydrophobic domain relativeto a constant polar head group affords compounds which exhibittransfection activity to varying degrees.

Transfection or membrane penetration is demonstrated by incorporatingthe subject diamines into various liposome/DNA complexes and exposingcells under desired conditions to initiate transfection. As seen in FIG.1, with effectiveness determined by luciferase light emissions (theluciferase plasmid pCMVL, see below, was utilized in a standard mannerto detect transfection levels), a serum-free 50:50 DOPE:PolyGum SV veryefficiently, as compared with DNA only, mediated DNA transfection of NIH3T3 cells.

The structural nature of the transfection vesicle influences theefficiency of the transfection. FIG. 2 clearly indicates that with a50:50 DOPE:PolyGum formulation, the SVs are much more capabletransfection carriers.

The effects of serum on PolyGum (MLV) mediated transfection of NIH 3T3cells is illustrated in FIG. 3. Under these transfection conditions,greatly increased transfection is found with serum-free conditions thanin the presence of 10% calf serum. Under other transfection conditions,little change is noted with serum transfection.

FIG. 4 plainly demonstrates how transfection of cells is influenced,with and without serum, by the side chain characteristics of thephosphatidylethanolamine utilized to generate the vesicles. The 50:50PolyGum:DOPE formulation is superior for serum-free transfection, whilethe 50:50 PolyGum:DMPE formulation prevails in the serum added case.

As the mole ratio of DOPE to Polygum in SVs is increased (See FIG. 5),the efficiency of transfection is lowered in a generally linear fashion.

For PolyGum containing SVs, FIG. 6 shows the DNA charge ratiooptimization data. For the ranges presented, clearly, a 2:1 PolyGum:DNAphosphate ratio maximizes transfection.

As seen in FIGS. 7 and 8, the presence of 2% FBS alters, as comparedwith serum-free conditions, the transfection levels for differentformulations. Without serum, and at a 2:1 lipid to DNA phosphate ratio,the 50:50 DOPE:PolyGum (SV) formulation is most efficient fortransfection. With serum, at the 2:1 ratio, the 50:50 DOPE:POHME (MLV)formulation is most efficient, however, the 50:50 DOPE:PolyGum speciesis second highest in efficiency of transfection. At the 4:1 ratio withserum the 50:50 DOPE:PolyGum (SV) formulation is once again the mostefficient transfection agent, with the MLV 50:50 DOPE:PolyGumcomposition in second. Distinctly, the PolyGum reagent is a useful agentfor transfecting cells.

FIG. 9 presents optimization data for methoxy PolyGum derivatives inwhich the type of hydrophobic side chain in altered. DP-PG-Me is mosteffective in transfection, while the DL-PG-Me derivative is leasteffective in transfection. It is noted that the addition of the methoxygroup appears to increase transfection, as long as the hydrophobic sidechain is of a suitable type. Compairing DM-PG-OH with DM-PG-Meillustrates that, given identical hydrobhobic side chains, the methoxyform is more efficient in transfection than the non-methoxy form.

Toxicity of the subject compounds was evaluated by application of thestandard Alamar Blue toxicity procedure. The results indicate lesstoxicity for both PolyGum MLV and SV formulations than for LIPOFECTIN™and several other amine containing vesicles.

EXAMPLES Example 1 Chemicals

Dioleoylphosphatidylethanolamine was purchased from Avanti Polar LipidsInc. (Birmingham, Ala.). Lipofectamine was obtained from LifeTechnologies. A liposome preparation containing DOTAP was obtained fromBoehringer Mannheim. Cholesterol was purchased from Sigma ChemicalCompany (St. Louis, Mo.). Alamar blue was obtained from AlamarBiosciences (Sacramento, Calif.). 2-hydroxyethylmethylamine,di(2-hydroxyethyl)methylamine, 2-methoxyethylmethylamine, anddi(2-methoxyethyl)methylamine starting materials are all available fromAldrich Chemical Company.

Example 2 Synthesis of (±)-2,3-Dihydroxy-1,4-N,N'-bis(2-tert-butyldiphenylsilyloxyethyl)-N,N'-dimethyl!butanediamine(See Compound 3 in Specific Scheme Above)

To a mixture of (±)-1,3-butadiene diepoxide (see compound 2 in SpecificScheme above, which is available from Aldrich Chemical Company) (0.93 g,12.0 mmol) and lithium perchlorate (5.09 g, 47.8 mmol) in absoluteethanol (50 mL) was addedN-methyl-2-(tert-butyldiphenylsilyloxy)ethylamine (Prepared according tothe procedure in: Chaudhary, S. K.; Hernandez, O. Tetrahedron Lett.1979, 99.) (see compound 1 in Specific Scheme above) (15.0 g, 47.8mmol). The reaction mixture was warmed to 60° C. and allowed to stir for24 hr. After this time, the reaction solution was allowed to cool toroom temperature and then transferred to a separatory funnel containingEt₂ O (75 mL). The resultant mixture was washed with saturated aqueousNaHCO₃. The organic layer was separated and subsequently washed with H₂O and brine, and then dried (Na₂ SO₄). The drying agent was filtered andthe filtrate was concentrated by rotary evaporation to give the crudeproduct as a yellow oil. Purification was accomplished by SiO₂ columnchromatography (3% MeOH in CH₂ Cl₂) to afford 6.96 g (81%) of compound 3(in above Specific Scheme) as an oil.

R_(f) =0.44 (10:90 methanol:dichloromethane); ¹ H NMR (300 MHz, CDCl₃) δ7.67 (m, 8H), 7.39 (m, 12H), 3.74 (t, J=6 Hz, 4H), 3.64 (m, 2H),2.73-2.58 (m, 6H), 2.53 (dd, J=4, 13, 2H), 2.32 (s, 6H), 1.04 (s, 18H);¹³ C NMR (75 MHz, CDCl₃) δ 135.3, 133.3, 129.6, 127.6, 68.6, 61.5, 60.7,59.6, 42.9, 26.7, 19.0; IR (KBr) 3420, 2931, 1112 cm⁻¹.

Example 3 Synthesis of (±)-2,3-Dioleoyloxy-1,4-N,N'-bis(2-tert-butyldiphenylsilyloxyethyl)-N,N'-dimethyl!butanediamine(See Compound 4 in Specific Scheme Above)

To a mixture of diamine compound 3 (4.26 g, 5.97 mmol), triethylamine(1.83 mL, 13.1 mmol), and 4-dimethylaminopyridine (0.146 g, 1.19 mmol)in CH₂ Cl₂ (30 mL) at 0° C. was added dropwise oleoyl chloride (3.954 g,13.14 mmol). On complete addition, the reaction mixture was allowed tostir at 0° C. for 4 hr. whereupon an additional portion of CH₂ Cl₂ (20mL) was added. The reaction mixture was then transferred to a separatoryfunnel and the organic layer was washed successively with saturatedaqueous NaHCO₃, H₂ O, and brine. The organic layer was dried (Na₂ SO₄),filtered, and the filtrate solvent removed in vacuo. The crude productso obtained was purified by SiO₂ column chromatography (1% MeOH in CH₂Cl₂) to yield 4.21 g (57%) of compound 4 as an oil.

R_(f) =0.31 (1:99 methanol:dichloromethane); ¹ H NMR (300 MHz, CDCl₃) δ7.66 (m, 8H), 5.35 (m, 4H), 5.15 (m, 2H), 3.68 (t, J=6 Hz, 4H), 2.59 (t,J=6 Hz, 4H), 2.49 (m, 4H), 2.27-2.22 (m, 10H), 2.01 (m, 8H), 1.28 (m,48H), 1.04 (s, 18H), 0.89 (t, J=7 Hz, 6H); ¹³ C NMR (75 MHz, CDCl₃) δ172.8, 135.4, 133.6, 129.9, 129.8, 129.6 (2), 129.4, 128.5, 127.5, 70.0,62.2, 59.7 (2), 58.1, 43.0, 34.2, 31.8, 29.7, 29.4, 29.3, 29.2, 29.1,29.0, 27.1 (2), 26.7, 24.9, 22.6, 19.0, 14.0; IR (KBr) 2927, 1733.2,1112 cm⁻¹.

Example 4 Synthesis of (±)-2,3-Dioleoyloxy-1,4-N,N'-bis(2-hydroxyethyl)-N,N'-dimethyl!butanediamine (See Compound 5 inSpecific Scheme Above)

To a solution of diamine compound 4 (4.21 g, 3.39 mmol) in THF (10 mL)at 0° C. was added dropwise a solution of tetrabutylammonium fluoride(20.4 mL of a 1M solution in THF, 20.4 mmol). The reaction was stirredat 0° C. for 15 h at which time analysis by thin layer chromatographyrevealed that no starting material was present. The reaction mixture wasdiluted with CH₂ Cl₂ (20 mL) and quenched by addition of saturatedaqueous NaHCO₃ (50 mL). The organic layer was separated and washedsuccessively with H₂ O and brine, and dried (Na₂ SO₄). After filtration,the organic layer was concentrated by rotary evaporation to give thecrude product as a yellow oil. The crude product was passed through ashort column of silica gel using 5% MeOH in CH₂ Cl₂ as the eluent toobtain a mixture of products. A second chromatographic step using SiO₂column chromatography (5% MeOH in CH₂ Cl₂) afforded 2.00 g (77%) of 5 asan oil.

R_(f) =0.48 (5:95 methanol:dichloromethane); ¹ H NMR (300 MHz, CDCl₃) δ5.33 (m, 6H), 5.62 (s, 2H), 3.55 (t, J=5 Hz, 4H), 2.61-2.50 (m, 8H),2.37 (t, J=8 Hz, 4H), 2.28 (s, 6H), 1.98 (m, 8H), 1.63 (m, 4H), 1.28 (m,48H), 0.87 (t, J=5 Hz, 6H); ¹³ C NMR (75 MHz, CDCl₃) δ 173.3, 129.9,129.7 (3), 129.5, 129.4, 69.5, 69.4, 59.7, 59.6, 59.5 (2), 58.6, 57.8,42.3, 34.2, 34.0, 31.8, 29.6 (2), 29.4, 29.2 (2), 29.1, 29.0, 27.1,27.0, 26.8, 24.8, 22.5, 22.4, 14.0, 13.9; IR (KBr) 3447, 2925, 1739,1512 cm⁻¹.

Example 5(±)-N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediaminiumiodide (See Compound 6, Which is PolyGum, in Specific Scheme Above)

A 25 mL round bottom flask was charged with diaminodiol compound 5 (1.26g, 1.65 mmol) and methyl iodide (16.5 mL, 265 mmol). The solution wasstirred at ambient temperature for 24 hr. After this time, the methyliodide was evaporated with the assistance of a steady stream of nitrogengas (note: this operation must be performed in a fume hood). The residueobtained on removal of the methyl iodide was doubly recrystallized fromacetonitrile to give 0.40 g (23%) of compound 6 as a white powder (mp169°-171° C.).

R_(f) =0.28 (5:95 methanol:dichloromethane); ¹ H NMR (300 MHz, CDCl₃) δ5.60 (m, 2H), 5.33 (m, 4H), 4.53 (m, 8H), 3.47 (s, 6H), 3.42 (s, 6H),2.50 (m, 4H), 2.00 (m, 8H), 1.65 (m, 4H), 1.29 (m, 44H), 0.88 (t, 6H);IR (KBr) 3355, 2901, 1753, 1467 cm⁻¹. The analytical calculation for C₄₈H₉₄ I₂ N₂ O₆ is: C=54.96; H=9.03; and N=2.67 and the found amounts were:C=54.78; H=9.04; and N=2.63.

Example 6 Synthesis of Other Subject Compounds

By analogous procedures to those for the oleoyl derivative in Example 3above, the various hydrophobic side chain derivatives were synthesized.Likewise, the derivatives made from the di(2-hydroxyethyl)methylamine,2-methoxyethylmethylamine, and di(2-methoxyethyl)methylamine startingmaterials were synthesized in an analogous manner to the steps presentedin Examples 1-5 for the silane blocked 2-hydroxyethylmethylamine, exceptthat for the 2-methoxyethylmethylamine, anddi(2-methoxyethyl)methylamine derived materials no introduction orremoval of the silane blocking groups was required (see specific schemesfor Compounds B and D above).

Example 7 Tissue Culture and Plasmids

NIH 3T3 cells were grown in Dulbecco's modified Eagles medium (DMEM)+10%fetal calf serum. The DNA plasmid pCMVL was prepared by standard methodsand used as a 369 ng/μl solution in TE, pH=7.6. The luciferase reporterplasmid pCMVL was prepared at UC Davis, and consists of the P. pyralisluciferase cDNA subcloned into the plasmid pRc/CMV (Invitrogen).

Example 8 Formation of MLVs and SVs

Multilamellar and small sonicated vesicles were prepared by addition ofthe cationic lipid DOHME together with DOPE, both as solutions inchloroform, to a 5 mL sample vial. The chloroform was removed via rotaryevaporation with the water bath set at a constant temperature of 37° C.The resulting thin lipid films were placed under high vacuum overnightto insure that all traces of solvent had been removed. The lipid mixturewas resuspended using distilled water (2 mmole total lipid/1 mL water)and vortex mixed to give a suspension of MLVs. This cloudy suspensionwas sonicated for fifteen minutes using a bath sonicator until a clearsuspension containing SVs was obtained.

Example 9 Formation of Liposome/DNA Complexes

Sequential addition of DMEM (with or without 10% fetal calf serum),pCMVL plasmid (for n=4, 4 μg), and liposome formulation into a 2 mLEppendorf tube gave a total volume of 800 μl. The relative amount ofliposome:pCMVL plasmid used was determined by the desired cationiclipid:DNA phosphate charge ratio. The mixing of these substances wasfollowed by thorough vortexing.

Example 10 Transfection of NIH 3T3 Cells

Usually, 24 well tissue culture plates containing 5.0×10⁴ cells/wellrapidly dividing adherent NIH 3T3 cells per well were transfected. Thegrowth media was removed via aspiration and the cells were washed oncewith 0.5 mL PBS/well. A 200 μl aliquot of liposome-DNA complex was addedto each well and the cells were allowed to incubate for 4 hr. at 37° C.If desired, at this time, 1 mL of DMEM+10% fetal calf serum/well wasadded and the cells were allowed to incubate for an additional 48 hr,after which assays of toxicity and/or efficacy were performed.Equivalent procedures were utilized for the DU-145 cells.

Example 11 Determination of Relative Luciferase Light Emissions

Transfection activity was measured using the luciferase assay.Luciferase assays were performed with a luciferase assay kit (purchasedfrom Analytical Luminescence Laboratories) using a Monolight 2010luminometer (Analytical Luminescence Laboratories, San Diego, Calif.)according to the manufacturer's instructions. The media was removed fromthe transfected cells via aspiration. 0.5 mL of luciferase buffer/wellwas added, and the cells were placed on ice for 15 min. Luciferase lightemissions from 100 μl of the lysate were measured using the luminometer.

Example 12 Optimization of Hydrophobic Side Chains

Shown in FIG. 9 are hydrophobic and polar domain transfection resultsfor various synthesized PolyGum derivatives. In the tests, 1.0×10⁵ NIH3T3 fibroblasts were plated 24 hours prior to transfection. Cell lysateswere obtained 48 hours after transfection and assayed for luciferasespecific activity. The data presented in FIG. 9 corresponds to theability of the subject cationic lipid to mediated delivery of 1microgram of plasmid DNA (pGL3I+).

Example 13 Alamar Blue Toxicity Assay

Alamar blue (Alamar Biosciences, Sacramento, Calif.), was diluted incell culture media to 10%, added to cells, and incubated for up to twohours. Reduced dye was quantitated using a CytoFluor 2300 fluorescenceplate reader with a 530 nm excitation filter and a 590 nm emissionfilter. Values expressed represent fluorescence less background. Thesame transfected cells can subsequently be assayed for reporter proteinactivity after Alamar Blue analysis.

The invention has now been explained with reference to specificembodiments. Other embodiments will be suggested to those of ordinaryskill in the appropriate art upon review of the present specification.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A compound having the structure: ##STR24##wherein n=0-10;R₂ is an acyl group; R₃ is an alkyl group, an alkenylgroup, or a hydroxylated alkyl or alkenyl group; and X⁻ is an anion. 2.A compound according to claim 1, wherein n is between 0 and three and R₃is an alkyl group of from 1 to 10 carbons.
 3. A compound according toclaim 1, wherein n is between 0 and 3 and R₃ is a methyl group.
 4. Acompound having the structure: ##STR25## wherein n=0-10;R₁ is an alkylgroup, an alkenyl group, or a hydroxylated alkyl or alkenyl group; R₂ isan acyl group; R₃ is an alkyl group, an alkenyl group, or a hydroxylatedalkyl or alkenyl group; R₅ is an alkyl or alkenyl group from 1 to 10carbons; and X⁻ is an anion.
 5. A compound according to claim 4, whereinn is between 0 and three, R₁ is an alkyl group of from 1 to 10 carbons,R₃ is an alkyl group of from 1 to 10 carbons, and R₅ is an alkyl oralkenyl group from 1 to 10 carbons.
 6. A compound according to claim 4,wherein n is between 0 and 3, R₁ is a methyl group, R₃ is a methylgroup, and R₅ is a methyl group.
 7. A compound according to claim 4,wherein n is 1, R₁ is a methyl group, R₃ is a methyl group, and R₅ is amethyl group.
 8. A compound having the structure: ##STR26## whereinn=0-10;R₂ is an acyl group; R₃ is an alkyl group, an alkenyl group, or ahydroxylated alkyl or alkenyl group; R₅ is an alkyl or alkenyl groupfrom 1 to 10 carbons; R₆ is an alkyl or alkenyl group from 1 to 10carbons; and X⁻ is an anion.
 9. A compound according to claim 8, whereinn is between 0 and three, R₃ is an alkyl group of from 1 to 10 carbons,R₅ is an alkyl or alkenyl group from 1 to 10 carbons, and R₆ is an alkylor alkenyl group from 1 to 10 carbons.
 10. A compound according to claim8, wherein n is between 0 and 3, R₃ is a methyl group, R₅ is a methylgroup, and R₆ is a methyl group.
 11. A compound according to claim 8,wherein n is 1, R₃ is a methyl group, R₅ is a methyl group, and R₆ is amethyl group.
 12. A compound having the structure: ##STR27## wherein Ris an alkyl or alkenyl group andX⁻ is an anion.
 13. A compound accordingto claim 12, wherein X⁻ is a halide.
 14. A compound having thestructure: ##STR28## wherein R is an alkyl or alkenyl group andX⁻ is ananion.
 15. A compound according to claim 14, wherein X⁻ is a halide. 16.A compound having the structure: ##STR29## wherein R is an alkyl oralkenyl group andX⁻ is an anion.
 17. A compound according to claim 16,wherein X⁻ is a halide.
 18. A compound having the structure: ##STR30##wherein n=0-10;R₁ is an alkyl group, an alkenyl group, or a hydroxylatedalkyl or alkenyl group; R₂ is an acyl group; R₃ is an alkyl group, analkenyl group, or a hydroxylated alkyl or alkenyl group; and X⁻ is ananion.
 19. A compound according to claim 18, wherein n is between 0 andthree, R₁ is an alkyl group of from 1 to 10 carbons, R₂ is an alkyl oralkenyl containing acyl group, and R₃ is an alkyl group of from 1 to 10carbons.
 20. A compound according to claim 18, wherein n is between 0and 3, R₁ is a methyl group, R₂ is an alkyl or alkenyl containing acylgroup, and R₃ is a methyl group.
 21. A compound according to claim 18,wherein n is 1, R₁ is a methyl group, R₂ is an alkyl or alkenylcontaining acyl group, and R₃ is a methyl group.
 22. A compound havingthe structure: ##STR31## wherein m=1-10;R₁ is an alkyl group, an alkenylgroup, a hydroxylated alkyl or alkenyl group, or an ether containingalkyl or alkenyl group; R₂ is an acyl group; R₃ is an alkyl group, analkenyl group, a hydroxylated alkyl or alkenyl group, or an ethercontaining alkyl or alkenyl group; R₄ is a hydroxylated alkyl or alkenylgroup, or an ether containing alkyl or alkenyl group; and X⁻ is ananion.